Protein kinases are a large group of intracellular and transmembrane signalling proteins in eukaryotic cells (Manning G. et al, (2002) Science, 298: 1912-1934). Phosphorylation of specific amino acid residues in target proteins by protein kinases can modulate their activity leading to profound changes in cellular signalling and metabolism. Kinases play key roles in the regulation of cellular proliferation, survival, differentiation and function. Many kinases have been implicated in disease and, as such, are attractive therapeutic targets.
The Tec family of kinases consists of Tyrosine kinase expressed in hepatocellular carcinoma (TEC), Interleukin-2 inducible T-cell kinase (ITK, also known as TSK and EMT), Resting lymphocyte kinase (RLK, also known as TXK), Bruton's tyrosine kinase (BTK), Bone marrow kinase on the X-chromosome (BMX, also known as ETK) (Bradshaw J M Cell Signal. 2010; 22(8):1175-84). These intracellular kinases play important roles in the development and function of lymphocytes and myeloid cells (Horwood et al. Int Rev Immunol. 2012; 31(2):87-103, Felices M et al. Adv Immunol. 2007; 93:145-84). Additionally, selected Tec family members such as ITK, TEC and BMX are expressed in cancerous cells where they may play a role in cancer cell survival and malignancy (Carson C C et al. Clin Cancer Res. 2015; 21(9):2167-76, Mano H. et al. Oncogene. 1990; 5(12):1781-6, Cenni B et al. Int Rev Immunol. 2012; 31(2):166-73).
ITK is an important component of T-cell signaling function and differentiation. ITK is activated upon stimulation of T-cell receptors and initiates a signaling cascade that results in cellular activation, cytokine release and rapid proliferation. ITK is important in T-helper (Th) cell development and function including Th1, Th2, Th9, Th17 and T-regulatory cell development (Fowell D J et al. 1999 Immunity 11:399-409; Gomez-Rodriguez J. et al. 2014 J. Exp Med 211:529-543, Gomez-Rodriguez J. et al 2016 Nat Commun. 2016; 7:10857). For example, ITK−/− CD4+ T-cells show significant reduction in the production of Th1 and Th17 cytokines and exhibit skewed T-effector/Treg-cell ratios with a bias towards FoxP3+ Treg (Kannan A et al 2015. J Neurosci. 35:221-233, Gomez-Rodriguez J. et al. 2014 J. Exp Med 211:529-543). Furthermore, specific inhibition of an allele-sensitive ITK mutant shows that ITK is important in Th1, Th2, Th17, and iNKT-cell cytokine production (Kannan A et al Eur. J. Immunol. 2015. 45: 2276-2285). Consequently, ITK is an important target for prevention or treatment of diseases involving Th cytokines, or where modulation of immunosuppressive Treg cells is desired. Furthermore, polymorphisms in the ITK promoter that increase ITK expression in humans have been linked to increased asthma incidence (Lee, S. H. et al. 2011 Ann Hum Genet 75:359-369) and ITK preferentially regulates the secretion of the Th2 cytokines IL-5 and IL-13 in models of allergic asthma suggesting that ITK inhibitors may be useful in the treatment of asthma (Muller C et al. 2003J Immunol. 170:5056-63). Also, ITK is upregulated in lesional skin from patients with allergic contact dermatitis, atopic dermatitis and psoriasis (von Bonin A et al. 2010. Exp. Derm; 20, 41-47).
RLK (TXK) is another Tec family member that is expressed in T-cells (Hu Q et al. 1995 J. Biol Chem. 270:1928-1934). TXK and ITK regulate Th cell-mediated responses via their differential expression in Th1 and Th2 cells, respectively (Sahu N et al. J. Immunol. 2008, 181:6125-6131). Furthermore, while ITK−/− mice have impaired in NKT cell generation this defect is exacerbated in the absence of both RLK and ITK (Felices M. et al. 2008, J Immunol. 180:3007-3018). Increased expression of RLK has been reported in patients with Behcet's disease, an inflammatory disorder associated with increased inflammation and Th1 cytokine production (Suzuki N et al. 2006 Clin Med Res. 4:147-151). Knockout of both ITK and RLK produces stronger effects on T-cell function than knockout of either kinase alone (Schaeffer et al. 1999 Science 284:638-641; Felices et al. 2008 J. Immunol. 180:3007-3018).
TEC kinase, first shown to be expressed in hepatocellular carcinoma (Mano et al. 1990 Oncogene. 5:1781-6), is expressed in normal B and T-cells and is up-regulated upon T-cell activation in Th1 and Th2 cells (Tomlinson M G et al 2004 Mol. Cell. Biol., 24:2455-2466). TEC may have different roles from either ITK or RLK. TEC has a unique subcellular distribution differential protein interactions compared with ITK and RLK (Tomlinson M G et al 2004 Mol. Cell. Biol., 24:2455-2466) and TEC, but not RLK or LTK, is a tyrosine kinase of c-Maf leading to enhancement of c-Maf-dependent IL-4 promoter activity (Liu C C et al. 2015 PLoS One. 10:e0127617). Lastly, TEC controls assembly of the non-cannonical caspase 8 inflammasome involved in fungal sepsis and Tec-deficient mice are highly resistant to candidiasis (Zwolanek F et al. 2014 PLoS Pathog 10, e1004525).
Experimental data using Tec-kinase family null animals supports the therapeutic benefit of kinase inhibition in human disease. ITK modulates neuroinflammation due to experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). ITK−/− mice exhibit reduced disease severity, and transfer of ITK−/−CD4+ T-cells into T-cell-deficient mice results in lower EAE disease severity (Kannan Ak et al. J. Neurosci, 2015; 35:221-233). ITK−/− mice exhibit decreased inflammatory response in contact hypersensitivity models (Von Bonin et al. Experimental Dermatology, 2010; 20, 41-47) and secretion of the Th2 cytokines IL-5 and IL-13 is decreased in models of allergic asthma in ITK−/− mice (Mueller C et al. J Immunol. 2003; 170(10):5056-63).
Data obtained with inhibitors of select Tec family kinases suggests that inhibitors of these kinases may be useful in the treatment of disease. Inhibitors of ITK, RLK and other Tec family members may be useful in the prevention or treatment of T-cell related diseases such as multiple sclerosis, asthma, atopic dermatitis, psoriasis and inflammatory bowel diseases as well as viral infections. For example, a small molecule inhibitor of ITK and RLK has shown efficacy in the mouse adoptive T-cell transfer model of colitis (Cho H-S et al. 2015; J. Immunol. 195: 4822-31).
Also, a selective ITK inhibitor blocked leukocyte lung infiltration following ovalbumin challenge in a rat model of asthma (Lin T A et al. 2004 Biochemistry. 43:11056-11062). Additionally, an ITK inhibitor was effective in mouse models of skin contact hypersensitivity (von Bonin A et al. 2010. Exp. Derm; 20, 41-47). Furthermore, ITK inhibitors can alter the HIV replication at various stages of viral life cycle including viral entry, gp120-induced actin reorganization, transcription from viral long terminal repeats (LTR) and virion assembly release from T-cells (Readinger J A et al. Proc Natl Acad Sci USA. 2008; 105(18):6684-9). Similarly ITK inhibition alleviates T-cell activation and murine myocardial inflammation associated with Coxsackie virus CVB3 infection (He F et al. Mol Immunol. 2014; 59(1):30-8) and ITK is required for efficient replication of influenza virus in infected T-cells (Fan K et al. J Gen Virol. 2012; 93(Pt 5):987-97). These data suggest that inhibitors of the Tec family kinases may be useful in the treatment of a variety of human and animal diseases.